Numerous assays are known for detecting analytes in a sample. Two general types of analytes are protein analytes and nucleic acid analytes. The technology and assays directed at detecting proteins have historically developed separately and largely independently from the technology and assays directed at detecting nucleic acids. Several reasons exist for this trend in these two fields. Initially, proteins and nucleic acids are chemically distinct and have very different chemical and physical properties. Assays to detect proteins were developed first, due in part to the presence and stability of proteins in the blood, urine, saliva, etc., samples which are readily available, and to the early correlation of physiological condition or disease with the presence proteins. Nucleic acids are generally less stable under assay conditions and are not found readily in free form in body fluids. Assays to detect nucleic acid analytes were developed much later and substantially independently from the protein assays. Assays for nucleic acids have been cumbersome, with low though-put, poor specificity and poor quantitative ability. Currently, protein analytical assays and nucleic acid analytical assays are considered to be two separate fields and practitioners in the two fields do not look to the literature of the other field for guidance in solving problems.
Early protein assays relied on the ability of antibodies to bind to specific protein analytes with sufficiently low dissociation constant (Kd) and with adequate specificity. Antibody capture assays are an easy and convenient screening method. In an antibody capture assay, an antigen is bound to a solid substrate, antibodies are allowed to bind to the antigen and then unbound antibodies are removed by washing. The bound antibodies are then detected using a detector molecule which specifically recognizes the antibody. Most antibody capture assays rely on an indirect method of detecting the antibody. For example, where the antibody is a murine antibody, the detector molecule might be a rabbit anti-mouse antibody which has been labeled with a detectable tag. Conventionally detectable tags have included radioactive isotopes, dyes and enzymes which act on a substrate to produce a detectable molecule, e.g., a chromogen.
In an antigen capture assay, the detection method identifies the presence of an antigen in a sample. In these methods, an antibody is bound to a solid support initially and then the antigen is allowed to react with the antibody to form a complex and the complex is subsequently detected. Alternatively, an antibody-antigen complex may be formed prior to binding of the antibody to a solid phase followed by detection of the complex.
A well known immunoassay is the enzyme-linked immunosorbent assay (ELISA) which when introduced in 1971 started a revolution in diagnostic methods. Conventional ELISA technology is a sandwich assay in which two antibodies or binding proteins simultaneously bind to an antigen or analyte. (Burgess, 1988.) Typically, a capture antibody is bound to a surface and binds to the analyte or antigen in a sample to form an antibody:antigen complex. A detecting antibody capable of binding the antigen or analyte and coupled to an enzyme is then used to form a capture antibody:antigen:detecting antibody sandwich complex and the complex is detected by measuring enzymatic activity of the enzyme bound to the detecting antibody.
Although assays using antibodies are very useful, it is generally accepted that the detection limit of an assay is limited by the Kd of the antibody used as the capture molecule (Griswold, W. (1987) J. Immunoassay 8: 145-171; O'Connor, T., et al. (1995) Biochem. Soc. Trans. 23(2): 393S). In practice, the detection limit of these assays is approximately 1% of the capture antibody Kd. As the concentration of analyte decreases to this sensitivity limit, the low percentage of capture molecules with bound analyte is insufficient to produce a detectable signal to noise ratio. For this reason, antibody based assays using state of the art fluorimetric or chemiluminescent detection systems have a detection limit of about 1 pg/ml (10 e-14 M for an “average” protein of molecular weight 50,000 daltons). See also Tijssen, P., Practice and Theory of Enzyme Immunoassays in Laboratory Techniques in Biochemistry and Molecular Biology, vol. 15, ed. by Burdon, R. H. and van Knippenberg, P. H., Elsevier, N.Y., 1985, pp. 132-136.
The detection of nucleic acid analytes requires different technology. In the mid-1980's, research in DNA technology lead to a method in which DNA could be amplified through a repeated enzymatic amplification process (Saiki et al., 1985; Mullis et al., 1987). The process, later named the polymerase chain reaction (PCR), uses two complementary oligonucleotide sequences (called primers) that flank the region of interest (5′ to 3′). The enzymatic process began with a denaturation step in the presence of the primers, then the temperature was lowered to allow primer annealing and Klenow fragment DNA polymerase I was added to extend the primers. Through repeated denaturation, annealing and extension of the desired fragment, exponential amplification of the target DNA was achieved. Many improvements have been made to PCR, but one important change was the incorporation of a DNA polymerase from Thermus aquaticus (Taq), a thermophilic bacterium (Saiki et al., 1988). Taq polymerase is a thermostable polymerase and is nearly unaffected by the denaturation steps involved in PCR, an improvement over the previous system where Klenow DNA polymerase I would have to be added to the reaction periodically because the enzyme did not tolerate the denturation step and lost activity.
In this type of enzymatic reaction, under conditions that allow primers to anneal efficiently, an exponential accumulation or a doubling of the template occurs every cycle. Such amplification allows for extremely low detection levels, some claiming to be able to detect single amplicons in a background of many other DNA molecules (Lentz et al., 1997).
Conventional PCR amplification is not a quantitative detection method, however. During amplification, primer dimers and other extraneous nucleic acids are amplified together with the nucleic acid corresponding to the analyte. These impurities must be separated, usually with gel separation techniques, from the amplified product resulting in possible losses of material. Although methods are known in which the PCR product is measured in the log phase (Kellogg et al., 1990; Pang et al, 1990), these methods require that each sample have equal input amounts of nucleic acid and that each sample amplifies with identical efficiency, and are therefore, not suitable for routine sample analyses. To allow an amount of PCR product to form which is sufficient for later analysis and to avoid the difficulties noted above, quantitative competitive PCR amplification uses an internal control competitor and is stopped only after the log phase of product formation has been completed (Becker-Andre, 1991; Piatak et al., 1993a, b).
In one application of PCR as an amplification system (Sano et al., 1992), an immuno-PCR method was developed that linked a microplate assay for a specific analyte with the amplification power of PCR for detection. The method detected, but did not quantitate, Bovine Serum Albumin (BSA) passively absorbed to an immuno-assay plate. Using an antibody specific for BSA, then bridging a biotin-labeled reporter amplicon with a protein A-streptavidin fusion protein, the assay utilized PCR amplification to detect several hundred molecules of BSA by agarose gel analysis of the reporter amplicon. However, this method could not be applied to biological samples due to the absence of aa specific analyte capture molecule. Others have improved the method by substituting the protein A-streptavidin fusion protein, which was not widely available, with a biotinylated secondary antibody and a streptavidin bridge to bind the biotinylated reporter amplicon (Zhou et al., 1993). The addition of the five assay reagents plus washing, PCR amplification and detection resulted in an assay that was laborious and was subjected to both stoichiometric and disassociation complications (Hendrickson et al., 1995). Another improvement in this assay approach came with the development of a method to covalently link a reporter amplicon to the secondary antibody (Hendrickson et al., 1995; Hnatowich et al., 1996). The direct linkage of the amplicon decreased the reagents used in the assay and the stoichiometric and disassociation complications that can occur. However, these methods still require significant post-PCR manipulations, adding to increased labor and the very real possibility of laboratory contamination.
Sandwich immuno-PCR is a modification of the conventional ELISA format in which the detecting antibody is labeled with a DNA label, and is applicable to the analysis of biological samples. In an early format of an antibody sandwich immuno-PCR, primary antibody was immobilized to a plate and sequentially, the sample, biotinylated detecting antibody, streptavidin, and biotinylated DNA, were added. This format was later improved by the direct conjugation of the DNA to the antibody and replacement of the gel electrophoresis by using labeled primers to generated a PCR product that can be assayed by ELISA (Niemeyer et al., 1996). The amplification ability of PCR provides large amounts of the DNA label which can be detected by various methods, typically gel electrophoresis with conventional staining (T. Sano et al., 1992, Science, 258: 120-122). Replication of the antibody-borne DNA label using PCR provides enhanced sensitivity for antigen detection. Immuno-PCR techniques have been extended to the detection of multiple analytes (Joerger et al., 1995; Hendrickson, 1995). While immuno-PCR has provided sensitivities exceeding those of conventional ELISA, purification of the amplified product by gel electrophoresis requires substantial human manipulation and is, therefore, time-consuming. Further, the primers used in the PCR amplification step may dimerize and the dimers are amplified under the PCR conditions leading to side products which compete for PCR amplification. In addition, matrix nucleic acids and other contaminating nucleic acids may be present or introduced and will be amplified by PCR.
Using a primary capture antibody, immuno-PCR methods and reagents are similar to a direct sandwich antigen ELISA, the difference coming at the choice of the detection method. Immuno-PCR methods have been successful and claim to obtain attomole level of sensitivity in some cases, including the detection of the following analytes: tumor necrosis factor ∝ (Sanna et al., 1995), β-galactosidase (Hendrickson et al., 1995), human chorionic gonadotropin, human thyroid stimulating hormone, soluble murine T-cell receptor (Sperl et al., 1995), recombinant hepatitis B surface antigen (Miemeyer et al., 1995), α-human atrial natriuretic peptide (Numata et al., 1997) and β-glucuronidase (Chang et al., 1997).
In immuno-PCR, antigen concentrations are generally determined by post PCR analysis of the reporter amplicon by either gel electrophoresis or PCR-ELISA. Quantitation of the DNA label by analyzing the endpoint PCR product is prone to errors since the rate of product formation decreases after several cycles of logarithmic growth (Ferre, 1992; Raeymakers et al., 1995) and the post PCR sample handling may lead to laboratory contamination. In addition, these methods require multiple steps and washes, during which the antibody:antigen complex may dissociate (Tijssen, P., ibid.).
Another method for amplicon quantitation, e.g. quantitative competitive PCR, uses laser induced capillary electrophoresis techniques to assess fluorescent PCR products (Fasco et al., 1995; Williams et al., 1996). In the context of a immuno-PCR analysis, all of these amplicon quantitation techniques require significant post PCR analysis and induce the possibility of PCR product contamination of the laboratory for following assays because of the handling requirements. Furthermore, these techniques are only able to analyze end-point PCR, PCR that has been stopped at a fixed PCR cycle number (e.g. 25 cycles of PCR). This poses a problem in the dynamic range of amplicon quantitation because only some PCR reactions may be in the log phase of amplification; reactions with high amounts of template will have used all PCR reagents and stopped accumulating amplicon exponentially and reactions with low amounts of template might not have accumulated enough amplicon to be detectable. Therefore, this phenomenon limits the detection range of PCR and can limit the analyte detection range of immuno-PCR assays.
In a further development of PCR technology, real time quantitative PCR has been applied to nucleic acid analytes (Heid et al., 1996). In this method, PCR is used to amplify DNA in a sample in the presence of a nonextendable dual labeled fluorogenic hybridization probe. One fluorescent dye serves as a reporter and its emission spectra is quenched by the second fluorescent dye. The method uses the 5′ nuclease activity of Taq polymerase to cleave a hybridization probe during the extension phase of PCR. The nuclease degradation of the hybridization probe releases the quenching of the reporter dye resulting in an increase in peak emission from the reporter. The reactions are monitored in real time. Reverse transcriptase (RT)-real time PCR (RT-PCR) has also been described (Gibson et al., 1996). The Sequence Detection system (ABI Prism, ABD of Perkin Elmer, Foster City, Calif.) uses a 96-well thermal cycler that can monitor fluorescent spectra in each well continuously in the PCR reaction, therefore the accumulation of PCR product can be monitored in ‘real time’ without the risk of amplicon contamination of the laboratory.
The Sequence Detection system takes advantage of a fluorescence energy theory known as Förster-type energy transfer (Lakowicz et al., 1983). The PCR reaction contains a fluorescently dual-labeled non-extendible probe that binds to the specific target between the PCR primers (FIG. 1a). The probe commonly contains a FAM (6-carboxyfluorescein) on the 5′-end and a TAMRA (6-carboxy-tetramethylrhodamine) on the 3′-end. When the probe is intact, the FAM dye (reporter dye) fluorescence emission is quenched by the proximity of the TAMRA dye (quencher dye) through Förster-type energy transfer. As PCR cycling continues, amplicon is produced and the hybridized probe is cleaved by the use of a polymerase that contains the 5′-3′ nuclease activity which chews through the probe, hence the nickname ‘TaqMan®’ given to the machine. With the cleavage of the probe, the reporter dye is then physically separated from the quencher dye, resulting in an increase in FAM fluorescence because of decreased quenching by TAMRA. The system uses an argon ion laser for fluorescence excitation (488 nm) and a charge-coupled device (CCD) camera to monitor the PCR reactions and collect fluorescence emission over the range of 500 nm to 660 nm for all 96-wells (SDS User's Manual, 1998). Using a algorithm that takes into account the overlapping emission spectra of the dyes used on the machine, the raw fluorescence data can be determined for the reporter, quencher and passive internal reference (ROX, 6-carboxy-X-rhodamine) dyes. The reference dye is used to normalize cycle to cycle fluorescence variations in each well. The Sequence Detection application then calculates a normalized change in reporter fluorescence (ΔRn) as follows; ΔRn=(ΔRn+)−(ΔRn−), where the ΔRn+ is the ‘reporter's emission fluorescence’/‘passive internal reference fluorescence’ for that particular PCR cycle and ΔRn− is the ‘reporter's emission fluorescence’/passive internal reference ‘fluorescence’ for a predetermined background period of the PCR reaction (typically cycles 3-15). Plotting the ΔRn versus PCR cycle reveals an amplification plot that represents the accumulation of the amplicon in the PCR reaction and cleavage of the probe (FIG. 1b). Using the provided software, the threshold value is either set manually by the user (at a fixed ΔRn value) or calculated, typically at 10 standard deviations above the mean of the background period of PCR (ΔRn). The point on the amplification plot at which a sample's fluorescence intersects the threshold value is referred to as the Ct value (PCR Cycle threshold) for that sample. Relative amounts of PCR product are compared among PCR reactions using the calculated Ct value. Using the Sequence Detection system, DNA and RNA have been successfully used for quantitative PCR (Heid et al., 1996) and rt-PCR (Gibson et al., 1996).
Nucleic acids have also been used as detector molecules in assays. The idea of “in vitro genetics” has been used to describe the isolation of binding nucleic acid ligands (Szostak et al., 1992). In general, the method involves taking a pool of very diverse nucleic acid sequences (typically degenerate oligonucleotides), introducing these sequences to a target and separating the bound sequences from the unbound sequences. The separation of the bound sequences results in a new pool of oligonucleotides that have been maturated by their preference to interact with the target, a type of genetic selection performed on the lab bench.
Nucleic acid and protein interactions in the cell are not uncommon occurrences. It is known that nucleic acids can fold to form secondary and tertiary structures and that these structures are important for binding interactions with proteins (Wyatt et al., 1989). The maturation of nucleic acid-protein binding interactions has been examined in vitro by varying the sequence of nucleic acid ligands (Tuerk et al., 1990). A technique known as SELEX (Systematic Evolution of Ligands by EXponential enrichment) is used to isolate novel nucleic acid ligands to a target of choice. These ligands were referred to as aptamers. The Greek root ‘apta’, meaning “to fit”, suggests a method for which the nucleic acid may fold and fit into pockets on target molecules. See U.S. Pat. No. 5,652,107; U.S. Pat. No. 5,631,146; U.S. Pat. No. 5,688,670; U.S. Pat. No. 5,652,107; A. D. (Ellington et al., 1992; Ellington et al., 1990; Greene et al., 1991).
The initial development of SELEX focused on a known protein-RNA interaction between Bacteriophage T4-DNA polymerase and a mRNA translational repressor. A well-characterized hairpin that presented a specific eight nucleic acid loop region was involved in the interaction with the polymerase (Andrake et al., 1988). The eight bases in this loop region were completely randomized (48=65,536 fold complexity) by synthesis on a nucleic acid synthesizer. Specific PCR primer regions were designed to flank the entire sequence for ease in amplification. The primer region also incorporated a bacteriophage T7 promoter so RNA could be easily transcribed from the DNA template. The RNA library, containing the 8-base random region, was mixed with the polymerase and protein-RNA complexes were separated through selective binding to a nitrocellulose filter. The nitrocellulose filters have a higher affinity for proteins than for nucleic acids, therefore capturing the protein and it's associated RNA ligand. The bound RNA was then eluted and rt-PCR performed with the primer set described above. A new matured RNA pool could then be in vitro transcribed from the resulting T7-promoter containing DNA template, completing one cycle of the selection. With repeated cycles of the binding and separation procedure, the random RNA pool was eventually matured to contain primarily two sequences; one sequence that was identical to the natural ligand found to bind the polymerase, and the other contained a four base difference from the natural ligand.
The power of maturing nucleic acid ligand pools in the SELEX procedure involves separating ligand-target complexes from free nucleic acid sequences. In the selection described above, a membrane that has a higher affinity for protein than RNA was used to create a new matured pool biased for sequences that interact with the protein. Maturation of the selection pool is accelerated by creating competition among the diverse RNA ligands in the pool. By lowering the target concentration, a situation is created where the binding sites are limited. The competition for these binding sites promotes higher affinity ligand selection. An unfortunate problem in some selections is the maturation of non-specific ligands, or ligands that bind to the nitrocellulose filter or other material in the selection procedure. One method used to avoid such ligands involves the use of carrier nucleic acid that cannot be extended by the selection PCR primer set (such as tRNA). Other methods of selection involve alternative procedures to separate ligand-target complexes, such as; affinity column binding, gel-shift assays and immuno-assay capture.
The design of a nucleic acid library involves three main considerations; minimizing amplification artifacts (resulting from miss priming), amount of randomness and length of the random region (Conrad et al., 1996). In designing a PCR amplification system, primer design is important to optimize the amplification of the specific amplicon of choice and to minimize non-specific amplification of other products by miss priming (either to amplicons of non-interest or primer-primer annealing). Primer design is also important in aptamer library design because of the large number of PCR cycles that are performed. A typical SELEX round will include 12-25 PCR cycles and a SELEX selection might include as much as 15 rounds, resulting in over 200 cycles of PCR on the selection pool. It is clear that miss primed PCR artifacts will accumulate in the selection pool that is subjected to such a large amount of PCR cycling. The amount of randomness can play an important role in the selection library if the study protein has a known nucleic acid sequence (such as the T4-DNA polymerase selection above). These libraries can be completely randomized in certain regions (keeping other wild-type sequence intact for secondary structure), or the wild-type sequences can be “doped” to contain a higher percentage of natural bases and a lower percentage of random bases (e.g. 70% G's and 10% A, C or T). Finally, the length of the random region can be varied over a wide range when using proteins that have known nucleic acid interactions. When selecting aptamers with proteins that have no known natural nucleic acid ligands, completely randomized libraries can be used, although the length must be considered. In longer completely randomized pools, greater secondary structure can be obtained because more bases are available. In shorter randomized pools, simpler secondary structure is obtained but a greater representation of all sequence possibilities is achieved (because of the physical limitation in the amount of DNA/RNA that one can select for in the first round). Ellington et al, 1994.
Since the original SELEX experiments, many nucleic acid ligands have been selected to a large variety of targets. Many proteins that normally bind nucleic acids have been shown to be good candidates for these SELEX selections. The designs of such selections have ranged from randomizing only the known binding region (T4 DNA Polymerase) to selections on completely randomized libraries. Other examples include; bacteriophage R17 coat protein (Schneider et al., 1992), E. Coli rho factor (Schneider et al., 1993), E. Coli ribosomal protein S1 (Ringquist et al., 1995) (and other S1 containing proteins, such as 30S particles and Qβ replicase (Brown et al., 1995)), phenylalanyl-tRNA synthetase (Peterson et al., 1993; Peterson et al., 1994), autoimmune antibodies that recognize RNA (Tsai et al., 1992), E2F transcription factor (Ishizaki et al., 1996) and various HIV associated proteins (Tuerk et al., 1993a; Giver et al., 1993; Tuerk et al., 1993b; Allen et al., 1995).
Aptamer selections have also been performed with proteins that were not known to bind to nucleic acids. Thrombin was one of the first candidates and its highest affinity aptamers were shown to be able to block thrombin's ability to cleave fibrinogen to fibrin (Bock et al., 1992; Kubik et al., 1994). Selections have also been carried out on many classes of proteins, including; growth factors (nerve growth factor (Binkley et al., 1995), basic fibroblast growth factor (Jellinek et al., 1993) and vascular endothelial growth factor (Jellinek et al., 1994)), antibodies (antibodies that bind to nuclear antigens (Tsai et al., 1992), insulin receptor (Doudna et al., 1995) and IgE class antibodies (Wiegand et al., 1996)), small molecules (cyanocobalamin (Lorsch et al., 1994), theophylline (Jenison et al., 1994), ATP (Sassanfer et al., 1993), GDP/GMP (Connell et al., 1994), chloroaromatics (Bruno et al., 1997), S-adenosyl methionine (Burke et al., 1997) and a panel of dyes (Ellingtion et al., 1990)) and a variety of other proteins (human thyroid stimulating hormone (Lin et al., 1996), human elastase (Bless et al., 1997), L-selectin (Hicke et al., 1996), protein kinase C (Conrad et al., 1994), Taq DNA polymerase (Dang et al., 1996) and reverse transcriptases, including; AMV (Chen et al., 1994), MMLV (Chen et al., 1994) and FIV (Chen et al., 1996).
The aptamers to the various reverse transcriptases demonstrate the specificity they can acquire. These aptamers did not share sequence motifs, except for the fact that they seem to interact through a series of “A” ribonucleotide bases. When cross tested for binding, the specific ligands did not cross-react with the other evolutionarily closely related transcriptases (Chen et al., 1994; Chen et al., 1996). Another example of specificity involves the RNA ligands to protein kinase C βII form (Conrad et al., 1994). The highest affinity aptamers showed a greater affinity for the βII form (by 1 order of magnitude via IC50 binding curve) when binding was compared to the alternatively spliced βI form of protein kinase C, even though they have only a 23 residue difference at the protein level. These aptamers also showed no apparent inhibition to other protein kinase C isozymes (α and ε forms).
The fact that one can generate novel nucleic acid ligands to a large variety of proteins led to the use of aptamers as an alternative to monoclonal and polyclonal antibody production for therapeutic and diagnostic uses. Diagnostic approaches using aptamers in place of antibodies have been evaluated. Aptamers to DNA polymerases have been used in hot start PCR to detect low copy number of the desired amplicon (Lin et al., 1997). Using an aptamer to block polymerase activity at low temperatures in PCR minimizes artifactual amplification and increases PCR sensitivity. Aptamers have also been used as a tool in assay development. An aptamer to neutrophil elastase was fluorescein labeled and used in a flow cytometry assay to determine elastase concentrations (Davis et al., 1996). The same aptamer was also used in an in vivo diagnostic imaging model of an inflamed rat lung (Charlton et al., 1997). Another aptamer to reactive green 19 (RG19) was also fluorescein labeled and used in a semi-quantitative bioassay for RG19 (Kawazoe et al., 1997). An immuno-assay using an aptamer detection reagent was also developed using a fluorescein labeled aptamer to VEGF (Drolet et al., 1996). The indirect immunoassay format was used for the quantitation of VEGF protein using a fluorescent substrate detection system.
In the enzyme-linked oligonucleotide assay (ELONA), one or more of the antibody reagents is replaced with an oligonucleotide sequence which specifically binds to the antigen. A specifically binding oligonucleotide sequence can be obtained by the in vitro selection of nucleic acid molecules which specifically bind to a target molecule using, for example, the SELEX method developed by L. Gold et al. (See Drolet, 1996). U.S. Pat. No. 5,472,841; U.S. Pat. No. 5,580,737; U.S. Pat. No. 5,660,985; U.S. Pat. No. 5,683,867; U.S. Pat. No. 5,476,766; U.S. Pat. No. 5,496,938; U.S. Pat. No. 5,527,894; U.S. Pat. No. 5,595,877; U.S. Pat. No. 5,637,461; U.S. Pat. No. 5,696,248; U.S. Pat. No. 5,670,637; U.S. Pat. No. 5,654,151; U.S. Pat. No. 5,693,502; U.S. Pat. No. 5,668,264; U.S. Pat. No. 5,674,685; U.S. Pat. No. 5,712,375; U.S. Pat. No. 5,688,935; U.S. Pat. No. 5,705,337; U.S. Pat. No. 5,622,828; U.S. Pat. No. 5,641,629; U.S. Pat. No. 5,629,155; U.S. Pat. No. 5,686,592; U.S. Pat. No. 5,637,459; U.S. Pat. No. 5,503,978; U.S. Pat. No. 5,587,468; U.S. Pat. No. 5,637,682; U.S. Pat. No. 5,648,214; U.S. Pat. No. 5,567,588; U.S. Pat. No. 5,707,796; U.S. Pat. No. 5,635,615; etc. WO 96/40991 and WO 97/38134 describe enzyme-linked oligonucleotide assays in which the capture antibody or the detecting antibody of a sandwich assay is replaced with a nucleic acid ligand. Generally, detection of the antigen:capture molecule complex is accomplished using a conventional enzyme-linked detecting antibody. Labeling of the oligonucleotide with a reporter enzyme, however, requires additional chemical synthesis steps and additional labor, difficulties also associated with assays which use antibody reagents as described above.
WO 96/40991 and WO 97/38134 also mention an embodiment in which the detection system is PCR amplification of a nucleic acid ligand which is part of the capture molecule:target molecule:detector molecule complex. These references suggest that the PCR primers used for amplification may contain reporter molecules such as enzymes, biotins, etc. Simple PCR amplification of a nucleic acid ligand provides additional quantities of the ligand, but has the disadvantage of requiring further separation steps to distinguish between the amplified ligand of interest and amplified nucleic acid impurities and primer dimers. Traditional gel separation requires intensive manual labor. Further, replicate experiments are required for statistical analysis and require additional time and labor. These problems exist for both DNA ligands and RNA ligands used in these oligonucleotide assays. The use of labeled primers allows detection of the PCR product, but does not overcome the problems of impurity and primer dimer amplification and is, therefore, not quantitative.
Despite these advances, a need continues to exist for a diagnostic method having improved sensitivity, improved dynamic range and less human manipulation in order to more rapidly analyze samples for the presence of and for the amount of a target antigen. Moreover, assays that use antibodies as capture reagents have detection limits that are approximately 1% of the antibody Kd (1 μg/ml for the highest affinity antibodies); however, a need exists for more sensitive assays of therapeutic and diagnostic analytes.